Human granulocytic anaplasmosis is caused by a tick-transmitted Anaplasma phagocytophilum, an obligate intracellular bacterium that is the paradigm for microbial subversion of neutrophils because it directly reprograms host cells for improved bacterial fitness, and in doing so, causes significant disease. Over 14 years we focused on neutrophil-A. phagocytophilum interactions and altered functions; we discovered AnkA, the nucleomodulin effector protein that silences the host CYBB promoter allowing intracellular survival, epigenetic changes at CYBB and other genes, and defined molecular mechanisms by which AnkA binds and modulates promoter activity via histone deacetylase-1 (HDAC1) recruitment. In fact, infection and AnkA mediate genome- wide epigenetic changes, including DNA methylation and histone deacetylation that likely belie the coordinated transcriptional reprogramming. Such extensive coordination of the host epigenome by a prokaryote is unprecedented and could explain how small genome prokaryotes subvert large genome eukaryotes for parasitic or symbiotic life. The basis of these reprogramming events is the focus of this competing renewal application in 3 specific aims: 1) the molecular basis of HDAC-1 recruitment to A. phagocytophilum AnkA will be studied with AnkA mutations that abrogate host transcription and reduce pathogen survival; 2) AnkA binds AT-rich loci throughout the genome at nuclear matrix attachment regions (MARs). We hypothesize that AnkA organizes 3 dimensional chromatin structure by bringing together function-related genes in specific chromosomal territories at large genomic distances to coordinate reprogramming. Here we will show that AnkA alters higher order granulocyte chromatin organization to influence accessibility of DNA to gene regulatory components and coordinate global transcription. AnkA mutants with defective or reduced HDAC-1 recruitment will be used to discern abrogated transcription and granulocyte functions; 3) the basis for A. phagocytophilum fitness and pathogenicity relates to its ability to alter gene programs in cells impacting tissues and whole organisms, as in infected humans. Proving the in vivo functions of AnkA and its relationship to microbial fitness and pathogenicity is not readily possible in humans. Therefore, we will demonstrate that cis- (Aim 1) and trans-transcriptional regulation mediated by AnkA also occurs in vivo and impacts infected cell function and disease manifestations by validating the results obtained in vitro or ex vivo in human cell cultures, and by measuring how changes in transcriptional programs impact pathogen propagation and disease in a mouse model. These integrated aims will extend the understanding of prokaryotes whose metabolism is shared with their host and organize cis and trans epigenetic changes for global reprogramming of host functions. The results will provide new directions for investigation in other microbes, novel approaches for disease intervention, or leverage the evolutionary adaptations of this and other organisms to create custom approaches for manipulation of eukaryotic hosts in disease and health.